Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are composed of two identical heavy chains and two identical light chains, each containing variable and constant domains. The variable domains of the heavy and light chains combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of antibodies comprises two heavy chains covalently linked by a disulphide bond. Bach heavy chain is in turn attached to a light chain, again via a disulphide bond. This produces a substantially “Y”-shaped molecule. This is shown schematically in FIG. 1.
Heavy chains are the larger of the two types of chain found in antibodies, with typical molecular mass of 50,000-77,000 Da, compared with the smaller light chain (25,000 Da).
There are five main classes or class s or classes of heavy chain which are γ, α, μ, δ and ε which are the constituents heavy chains for: IgG, IgA, IgM, IgD and IgE respectively. IgG is the major immunoglobulin of normal human serum, accounting for 70-75% of the total immunoglobulin pool. This is the major antibody of secondary immune responses. It forms a single tetramer of two heavy chains plus two light chains.
IgM accounts for approximately 10% of the immunoglobulin pool. The molecules, together with J-chains, form a pentamer of five of the basic 4-chain structures. The individual heavy chains have a molecular weight of approximately 65,000 and the whole molecule has a molecular weight of about 970,000. IgM is largely confined to the intravascular pool and is the predominant early antibody.
IgA represents 15-20% of human serum immunoglobulin pool. More than 80% of IgA occurs as a monomer. However, some of the IgA (secretory IgA) exists as a dimeric form.
IgD accounts for less than 1% of the total plasma immunoglobulin.
IgE, although scarce in normal serum, is found on the surface membrane of basophils and mast-cells. It is associated with allergic diseases such as asthma and hay-fever.
In addition to the five main classs or classes, there are four subclasses for IgG (IgG1, IgG2, IgG3 and IgG4). Additionally there are two subclasses for IgA (IgA1 and IgA2).
There are two types of light chain: Lambda (λ) and Kappa (κ). There are approximately twice as many κ as λ molecules produced in humans, but this is quite different in some mammals. Each chain contains approximately 220 amino acids in a single polypeptide chain that is folded into one constant and one variable domain. Plasma cells produce one of the five heavy chain types together with either κ or λ molecules. There is normally approximately 40% excess free light chain production over heavy chain synthesis. Where the light chain molecules are not bound to heavy chain molecules, they are known as “free light chain molecules”. The κ light chains are usually found as monomers. The λ light chains tend to form dimers.
There are a number of proliferative diseases associated with antibody producing cells. FIG. 2 shows the development of B-cell lineage and associated diseases. These diseases are known as malignant plasma cell diseases. They are summarised in detail in the book “Serum-free Light Chain Analysis” A. R. Bradwell, available from The Binding Site Limited, Birmingham, UK (ISBN: 07044 24541).
In many such proliferative diseases a plasma cell proliferates to form a monoclonal tumour of identical plasma cells. This results in production of large amounts of identical immunoglobulins and is known as a monoclonal gammopathy.
Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% respectively of cancer deaths in the United Kingdom. Multiple myeloma is the second-most common form of haematological malignancy after non-Hodgkin lymphoma. In Caucasian populations the incidence is approximately 40 per million per year. Conventionally, the diagnosis of multiple myeloma is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine and related organ or tissue impairment such as hypercalcaemia, renal insufficiency, anaemia or bone lesions. Normal plasma cell content of the bone marrow is about 1%, while in multiple myeloma the content is typically greater than 30%, but may be over 90%.
AL amyloidosis is a protein conformation disorder characterised by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, these patients present with heart or renal failure but peripheral nerves and other organs may also be involved.
There are a number of other diseases which can be identified by the presence of monoclonal immunoglobulins within the blood stream, or indeed urine, of a patient. These include plasmacytoma and extramedullary plasmacytoma, a plasma cell tumour that arises outside the bone marrow and can occur in any organ. When present, the monoclonal protein is typically IgA. Multiple solitary plasmacytomas may occur with or without evidence of multiple myeloma. Waldenström's macroglobulinaemia is a low-grade lymphoproliferative disorder that is associated with the production of monoclonal IgM. There are approximately 1,500 new cases per year in the USA and 300 in the UK. Serum ISM quantification is important for both diagnosis and monitoring. B-cell non-Hodgkin lymphomas cause approximately 2.6% of all cancer deaths in the UK and monoclonal immunoglobulins have been identified in the serum of about 10-15% of patients using standard electrophoresis methods. Initial reports indicate that monoclonal free light chains can be detected in the urine of 60-70% of patients. In B-cell chronic lymphocytic leukaemia monoclonal proteins have been identified by free light chain immunoassay.
Additionally, there are so-called MGUS conditions. These are monoclonal gammopathy of undetermined significance. This term denotes the unexpected presence of a monoclonal intact immunoglobulin in individuals who have no evidence of multiple myeloma, AL amyloidosis, Waldenström's macroglobulinaemia, etc. MGUS may be found in 1% of the population over 50 years, 3% over 70 years and up to 10% over 80 years of age. Most of these are IgG- or IgM-related, although more rarely IgA-related or bi-clonal. Although most people with MGUS die from unrelated diseases, MGUS may transform into malignant monoclonal gammopathies.
In at least some cases for the diseases highlighted above, the diseases present abnormal concentrations of monoclonal immunoglobulins or free light chains. Where a disease produces the abnormal replication of a plasma cell, this often results in the production of more immunoglobulins by that type of cell as that “monoclone” multiplies and appears in the blood.
The identification of monoclonal immunoglobulins, and the heavy and light chains making up those immunoglobulins may be carried out in a number of ways. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) have been used for a number of years to identify the presence of monoclonal proteins in the serum. Serum protein electrophoresis is the standard method for screening for intact immunoglobulin multiple myeloma and is based upon scanning gels in which serum proteins have been separated, fixed and stained. There are limitations associated with this method, including that some samples from patients with myelomas appear normal by electrophoresis. This results in the possibility of missing patients and misdiagnosis of the disease. Furthermore, the technique does not readily allow for the accurate quantitative determination of the various proteins identified, particularly at low concentrations. Serum electrophoresis can be used to identify the presence of free light chains, but the detection limit is between 500 mg/L and 2,000 mg/L, depending upon whether or not the monoclonal protein migrates alongside β proteins. Sermon protein electrophoresis is negative for free light chains in all patients with non-secretory myeloma.
Immunofixation electrophoresis uses a precipitating antibody against the immunoglobulin molecules. Whilst this improves the sensitivity of the test it cannot be used to quantify monoclonal immunoglobulins because of the presence of the precipitating antibody. Immunofixation electrophoresis is also rather laborious to perform and interpretation may be difficult. Capillary zone electrophoresis is used in many clinical laboratories for serum protein separation and is able to detect most monoclonal immunoglobulins. However, when compared with immunofixation, capillary zone electrophoresis fails to detect monoclonal proteins in 5% of samples. These so-called “false negative” results encompass low-concentration monoclonal proteins.
Total κ and λ assays have been produced. However, total κ and total λ assays are too insensitive for the detection of monoclonal immunoglobulin or free light chain. This is due to high background concentrations of polyclonal bound light chains which interfere with such assays.
More recently, the applicants have developed a sensitive assay that can detect the free κ light chains and separately, the free λ light chains. This method uses a polyclonal antibody directed towards either the free κ or the free λ light chains. This is discussed in detail in the book by A. R. Bradwell. The possibility of raising such antibodies was also discussed as one of a number of different possible specificities, in WO 97/17372. This document discloses methods of tolerising an animal to allow it to produce desired antibodies that are more specific than prior art techniques could produce. The free light chain assay uses the antibodies to bind to free λ or free κ light chains. The concentration of the free light chains is determined by nephelometry or turbidimetry. This involves the addition of the test sample to a solution containing the appropriate antibody in a reaction vessel or cuvette. A beam of light is passed through the cuvette and as the antigen-antibody reaction proceeds, the light passing through the cuvette is scattered increasingly as insoluble immune complexes are formed. In nephelometry, the light scatter is monitored by measuring the light intensity at an angle away from the incident light, whilst in turbidimetry light scatter is monitored by measuring the decrease in intensity of the incident beam of light. A series of calibrators of known antigen (i.e. free κ or free λ) concentration are assayed initially to produce a calibration curve of measured light scatter versus antigen concentration.
This form of assay has been found to successfully detect free light chain concentrations. Furthermore, the sensitivity of the technique is very high.
Because a monoclonal plasma cell of the type causing e.g. multiple myeloma will produce only one type of antibody with a λ or a κ light chain, the relative ratio of λ or κ will change.
If the amount of the free λ light chain and the amount of the free κ light chain are known, it is possible to calculate the ratio between the free λ and the free κ light chains. An example of the results of plotting serum λ versus serum κ concentrations for patients with different diseases is shown in FIG. 3. The amount of free λ and free κ is skewed away from the normal concentrations because of the monoclonal nature of many of these diseases.
Measuring the κ:λ ratio for free light chains assists in the diagnosis of the disease. Furthermore, if the disease is treated, for example by chemotherapy or radiotherapy, the technique allows the disease to be monitored. If the disease is successfully being treated, then the concentrations of free light chains, which have a relatively short life span within the blood, will change and move more towards the normal concentrations observed for normal sera. Moreover, in malignant plasma cell diseases there is often suppression of production of the opposite light chain, so the κ:λ ratio can be more sensitive than individual FLC measurements.
Haraldsson A., et al. (Ann. Clin. Biochem (1991), 28(5): 461-466) discloses ELISA assays for the determination of kappa and lambda ratios within total IgG, IgA and IgM.
Chui S. A., et al. (J. Clin. Immunol. (1991), 11(4): 219-223) discloses studying patients with primary IgA nephropathy with an ELISA kit. The ELISA uses monoclonal mouse anti-human IgA1 as a solid phase capture and peroxidase-labelled anti-kappa and anti-lambda antibodies. IgA nephropathy is a kidney disease caused when IgA builds up as deposits in kidneys and appears to run in families.
FIG. 4 indicates that not all such diseases produce free light chains. The use of free light chains as a marker for the diseases is therefore not 100% successful.